Apparatus and method for preparative supercritical fluid chromatography

ABSTRACT

A fractionated sample collection process and device having at least one pressurized chamber used to gather and store liquid samples generated from a flow stream containing a mixture of highly compressed gas, compressible liquid, or supercritical fluid and a relatively incompressible liquid. A bank of multiple collection chambers is secured together in a frame to form a cassette unit. Each collection chamber may house a replaceable liner such as a test tube vial, to hold a fractionated liquid phase sample. After filling with sample, a collection chamber is returned to a clean state and ready for refilling with a new sample by manually or automatically replacing the liner.

This application claims the benefit of application serial No.60/154,038, Preparative Supercritical Fluid Chromatography, filed Sep.16, 1999.

BACKGROUND OF THE INVENTION

A substantial need exists for industries to recover purified componentsof interest from samples containing simple or complex mixtures ofcomponents. Many technologies have been developed to meet this need. Fordissolvable, nonvolatile components, the technology of choice has beenliquid elution chromatography.

Analysts have several objectives in employing preparative elutionchromatography. First, they wish to achieve the highest available purityof each component of interest. Second, they wish to recover the maximumamount of the components of interest. Third, they wish to processsequential, possibly unrelated samples as quickly as possible andwithout contamination from prior samples. Finally, it is frequentlydesirable to recover samples in a form that is rapidly convertibleeither to the pure, solvent-free component or to a solution of knowncomposition which may or may not include the original collectionsolvent.

In the case of normal phase chromatography, where only organic solventsor mixtures are used as eluants, typical fraction volumes of tens tohundreds of milliliters are common. The fraction must then be evaporatedover substantial time to recover the component residues of interest. Inreversed phase chromatography, where mixtures of organic solvents andwater are used as the elution mobile phase, a secondary problem arises.After removal of lower boiling solvents, recovered fractions mustundergo a water removal step lasting from overnight to several days.Thus, availability of the recovered components of interest is delayed byhours or days, even after the separation process is complete. Thislatter problem can create a serious bottleneck in the entirepurification process when enough samples are queued.

Where difficult separation conditions exist or separation speed is arequirement, a subset of elution chromatography, known as highperformance liquid chromatography (HPLC), is preferred. This HPLCtechnique is used both as an analytical means to identify individualcomponents and as a preparative means of purifying and collecting thesecomponents.

For analytical HPLC, samples with component levels in the nanogram tomicrogram range are typical. Preparative HPLC systems typically dealwith microgram to multiple gram quantities of components per separation.Preparative HPLC systems also require a means to collect and storeindividual fractions. This is commonly performed, either manually orautomatically, simply by diverting the system flow stream to a series ofopen containers.

Drawbacks exist to the current use of preparative HPLC. Elution periodsranging from several minutes to hours are necessary for each sample.Further, even in optimal conditions only a small fraction of the mobilephase contains components of interest. This can lead to very largevolumes of waste mobile phase being generated in normal operation of thesystem.

An alternative separation technology called supercritical fluidchromatography (SFC) has advanced over the past decade. SFC uses highlycompressible mobile phases, which typically employ carbon dioxide (CO2)as a principle component. In addition to CO2, the mobile phasefrequently contains an organic solvent modifier, which adjusts thepolarity of the mobile phase for optimum chromatographic performance.Since different components of a sample may require different levels oforganic modifier to elute rapidly, a common technique is to continuouslyvary the mobile phase composition by linearly increasing the organicmodifier content. This technique is called gradient elution.

SFC has been proven to have superior speed and resolving power comparedto traditional HPLC for analytical applications. This results from thedramatically improved diffusion rates of solutes in SFC mobile phasescompared to HPLC mobile phases. Separations have been accomplished asmuch as an order of magnitude faster using SFC instruments compared toHPLC instruments using the same chromatographic column. A key factor tooptimizing SFC separations is the ability to independently control flow,density and composition of the mobile phase over the course of theseparation.

SFC instruments used with gradient elution also reequillibrate much morerapidly than corresponding HPLC systems. As a result, they are ready forprocessing the next sample after a shorter period of time. A commongradient range for gradient SFC methods might occur in the range of 2%to 60% composition of the organic modifier.

It is worth noting that SFC instruments, while designed to operate inregions of temperature and pressure above the critical point of CO2, aretypically not restricted from operation well below the critical point.In this lower region, especially when organic modifiers are used,chromatographic behavior remains superior to traditional HPLC and oftencannot be distinguished from true supercritical operation.

In analytical SFC, once the separation has been performed and detected,the highly compressed mobile phase is directed through a decompressionstep to a flow stream. During decompression, the CO2 component of themobile phase is allowed to expand dramatically and revert to the gasphase. The expansion and subsequent phase change of the CO2 tends tohave a dramatic cooling effect on the waste stream components. If careis not taken, solid CO2, known as dry ice, may result and clog the wastestream. To prevent this occurrence, heat is typically added to the flowstream. At the low flow rates of typical analytical systems only a minoramount of heat is required.

While the CO2 component of the SFC mobile phase converts readily to agaseous state, moderately heated liquid organic modifiers typicallyremain in a liquid phase. In general, dissolved samples carried throughSFC system also remain dissolved in the liquid organic modifier phase.

The principle that simple decompression of the mobile phase in SFCseparates the stream into two fractions has great importance with regardto use of the technique in a preparative manner. Removal of the gaseousCO2 phase, which constitutes 50% to 95% of the mobile phase duringnormal operation, greatly reduces the liquid collection volume for eachcomponent and thereby reduces the post-chromatographic processingnecessary for recovery of separated components.

A second analytical purification technique similar to SFC issupercritical fluid extraction (SFE). Generally, in this technique, thegoal is to separate one or more components of interest from a solidmatrix. SFE is a bulk separation technique, which does not necessarilyattempt to separate individually the components, extracted form thesolid matrix. Typically, a secondary chromatographic step is required todetermine individual components. Nevertheless, SFE shares the commongoal with prep SFC of collecting and recovering dissolved components ofinterest from supercritical flow stream. As a result, a collectiondevice suitable for preparative SFC should also be suitable for SFEtechniques.

Expanding the technique of analytical SFC to allow preparative SFCrequires several adaptations to the instrument. First the systemrequires increased flow capacity. Flows ranging from 20 ml/min to 200ml/min are suitable for separation of multi-milligram up to gramquantities of materials. Also, a larger separation column is required.Finally, a collection system must be developed that will allow, at aminimum, collection of a single fraction of the flow stream whichcontains a substantially purified component of interest. In addition,there frequently exists a compelling economic incentive to allowmultiple fraction collections from a single extracted sample. Themodified system must also be able to be rapidly reinitialized eithermanually or automatically to allow subsequent sample injection followedby fraction collection.

Several commercial instances of preparative SFC instrumentation havebeen attempted which have employed different levels of technology tosolve the problems of collection. A representative sampling of theseproducts includes offerings from Gilson, Thar, Novasep, and ProChrome.However, no current implementation succeeds in providing high recovery,high purity, and low carryover from sample to sample. For example, onesystem may use the unsophisticated method of simply spraying thecollection stream directly into a large bottle, which results in highsample loss, presumably due to aerosol formation. Another system uses acyclonic separator to separate the two streams, but provides no rapid orautomated means of washing the separators to prevent carryover. Suchinstruments are typically employed to separate large quantities ofmaterial by repetitive injection so that no sample-to-sample cleaningstep is required. Other systems use a collection solvent to trap asample fraction into a volume of special solvent in a collectioncontainer. This technique uses relatively large quantities of hazardoussolvents to perform sample collection, is prone to sample fractionconcentration losses or degradation, and possible matrix interferencesexist between fractionated samples and collection solvent constituents.

An example of a SFC system is illustrated outside of the outlinedsection 10 in FIG. 1. The schematic flow diagram is a packed-columnsupercritical fluid chromatography (SFC) system from initial modifiersupply to a detector. The system has a carbon dioxide supply tank 200,line chiller 220, pump 202, modifier tank 204 and pump 206, dampener andpressure transducer 208, leading to a mixing column 210, connected to aninjection valve 212 that is connected to at least one packedchromatography column 214, and a detector 216.

In a SFC system, liquefied compressed carbon dioxide gas is suppliedfrom cylinders 200. High pressure tubing 218 connects the carbon dioxidereservoir tank 200 to the carbon dioxide pump 202. The tubing may becooled 220 prior to connecting to the pump 202. The system uses twoHPLC-type reciprocating pumps 202, 206. One pump 202 delivers carbondioxide and the other pump 206 delivers modifier 204, such as methanol.The carbon dioxide and modifier are combined, creating a mixture ofmodifier dissolved into the supercritical fluid.

The combined supercritical fluid is pumped at a controlled mass-flowrate from the mixing column 210 through transfer tubing to a fixed-loopinjector 212 where the sample of interest is injected into the flowsystem. The sample combines with the compressed modifier fluid insidethe injection valve 212 and discharges into at least one packedchromatography column 214. After fractionation of the sample occurs inthe columns 214, the elution mixture passes from the column outlet intoa detector 216.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a fractioncollection device for supercritical fluid flow systems.

It is a further object of the present invention to provide a device thatcollects fractionated components of sample solutes into one or morecollection containers.

The present invention relates to sample recovery after separation bysupercritical fluid chromatography or supercritical fluid extraction,and improvements therein.

More specifically, the present invention relates to optimally separatinga liquid phase, containing sample components of interest, from a muchlarger gaseous phase after the controlled expansion, or decompression,of a single chromatographic mobile phase from a high working pressure toa lower pressure where it is unstable. The controlled decompressioncauses a phase separation between liquid and gaseous phases while at thesame time aerosol formation is strongly suppressed within the transfertubing.

It is a further object of the present invention to provide a device andmethod to separate monophasic fluids that are mixtures of highlycompressed or liquefied gasses and organic liquid modifiers into gaseousand liquid phases inside transfer tubing prior to collection offractions of the liquid phase into one or more unique collectionchambers. The collection of fractions of the liquid phase intocollection chambers minimizes liquid solvent use and waste throughefficient gas and liquid phase separation prior to entering collectionchambers. The collection technique uses no additional solvents forcollection of fractions.

This invention provides a cassette bank of multiple chambers to collectand store separated or extracted fractions. Each collection cassetteincludes one or more collection chambers, and each chamber can receive apurified liquid fraction. Each chamber may hold a removable samplecollection liner. The collection liners may be individually removed,substituted, stored, cleaned and re-used, or discarded. One purpose ofthe collection liner is to provide a simplified means of transportingthe collected liquid fraction from the cassette. A second purpose of thecollection liner is to provide a means to eliminate cross-contaminationof consecutive samples by providing an easily replaceable,uncontaminated liner in each collection chamber for each sample.

The present invention manually or automatically controls one or morevalves and a sealing mechanism for collection chambers such thatmultiple liquid phase fractions from one sample may be collected intoone or more chambers without mechanically adjusting the collectionchamber seals. This method allows for rapid switching between collectionchambers in the event of closely separated peaks in the chromatograghicflow stream.

It is a further object of the present invention to facilitate a manualor automatic reset of the collection system to allow consecutive samplesto be processed in a rapid manner. Technical difficulties arise in theimplementation of a collection system that satisfies all the analystsobjectives stated above. The major problem centers around the tremendousexpansion (typically 500-fold) of the pressurized liquid orsupercritical CO2 fraction of the mobile phase that violently transformsinto a gas at atmospheric pressure. This transition has four majornegative effects with regard to liquid phase sample collection.

First, as mentioned above, the expanding CO2 causes a severe temperaturedrop that has the possibility of forming dry ice and clogging thesystem. Since flows of preparative SFC systems are much higher thancorresponding analytical systems, considerable more heat must be addedto compensate for the temperature drop. Care must be taken; however, notto allow the actual temperature to rise in the flow system since thismay cause damage to thermally unstable compounds of interest. Higherorganic modifier content reduces the severity of this problem, both byadding heat capacity and by dissolving the CO2, thereby preventing dryice formation.

Second, as the CO2 expands, it rapidly loses any solvating power it hadin the compressed state. If components of interest are largely dependenton the CO2 for solubility they will lose their primary means oftransport through the flow system. Solid components will accumulate andeventually clog the flow path causing system failure. Again, the organicmodifier component is an important factor here since the liquid willcontinue to solvate the components of interest and transport them to acollection device. Care must be taken not to introduce too much heatinto the flow stream as to drive the organic modified also into the gasphase, otherwise its beneficial effect of transporting the solutes willbe lost.

Third, it is beneficial to complete the transition from liquid togaseous CO2 in as short a period as possible after the initialdecompression stage. While in the liquid state, CO2 can disperse theorganic modifier containing components of interest even when it is notdense enough to have any significant solvating power. This dispersioncan have the effect of remixing components that had been efficientlyseparated by the SFC process prior to decompression. The faster the CO2can be converted the less chromatographic degradation can occur. Twofactors seem to predominate in controlling the ability to volatilize theliquid phase CO2: a) efficient heat transfer between the heat source andthe flowing liquid and b) residence time of the CO2 in the heatedregion. The first factor can be positively affected by selection of ahighly conductive material such as copper for heater fabrication.Insuring excellent thermal contact between the heater and a thin-walledtransfer tubing also facilitates heat transfer to the flowing fluids.Residence time of the decompressing fluid can be controlled by steppingthe pressure drop over a series of one or more restrictors in thetransfer line. Higher backpressure slows the linear velocity of thebiphasic fluid in the heater. So long as the back pressure generated bythese restrictions do not interfere with the SFC density regulation inthe high pressure separation region, a great deal of tunability ispossible for optimizing heat transfer.

Fourth, due to the expansion, linear velocities of the depressurizingfluid increase dramatically in the transfer tubing. Residual liquids ofthe system are moved along the flow path largely by shear forces fromthe expanding gas. This turbulent environment is ideal for the creationof aerosols, whereby very small droplets of modifier liquid areentrained in the gas phase as a “mist”. It is a finding of this studythat the aerosol formation within the transfer tubing can be almostcompletely controlled by proper temperature control of the expandingtwo-phase system. Aerosol formation is a greater problem at lowertemperatures. It is a surprising finding of this work that higher levelsof organic modifier with correspondingly lower CO2 content requirehigher temperature levels to prevent visible aerosol formation.

In the preferred exemplary embodiment, the SFC collection system iscomposed of a moderately restrictive, thermally regulated stainlesssteel transfer tube which extends from a back pressure regulationcomponent of the SFC chromatograph into a multi-port distribution valveand from the valve to a variety of flowpaths leading either throughdiscrete collection chambers or directly connected to a vented commonwaste container.

Initial separation of the liquid phase sample from carbon dioxide gasoccurs immediately at the point of initial decompression within thebackpressure regulator of the SFC or SFE instrument. By providingdownstream restriction, a minimum backpressure sufficient to prevent theformation of solid CO2 can be maintained while liquid CO2 is present inthe transfer lines.

The remainder of the CO2 evaporation and separation from the organicmodifier occurs in the stainless steel transfer tubing prior to enteringthe cassette. This is accomplished by exposing the transfer tubing to aseries of one or more heaters designed to optimize thermal transfer tothe fluid. Ideally, this heater series transfers sufficient energy tothe liquid CO2 portion of the emerging fluid to allow for completeevaporation of the liquid CO2 and raise the fluid temperaturesufficiently to prevent the transfer tubing from icing externally.Because rates of heat transfer are time dependent, it is beneficial toslow the velocity of fluids within the heater series.

During the CO2 evaporation process within the first heated zone,significant separation between the gaseous CO2 and liquid modifieroccurs. However, the separation to pure CO2 and pure organic modifier isnever realized for several reasons. First, some organic modifier istypically also evaporated into the gas state. The degree of evaporationis largely dependent on the absolute temperature of the fluids withinthe transfer tubing. While organic modifier evaporation does lead tolower recovery of liquid phase, it does not necessarily reduce therecovery of dissolved components of interest which do not typically havelow enough boiling points to convert to vapor. Second, a fraction of CO2will remain dissolved in the organic liquid. Both temperature andpressure determine the amount of residual CO2. Higher temperaturesreduce CO2 solubility while higher pressures increase CO2 solubility.

Aerosol formation of the liquid phase is a common problem in SFC samplecollection and is a primary cause of loss of the organic liquid phasethat contains the dissolved components of interest. Higher temperaturesreduce the aerosol generation. The composition of the separated phasesalso is a factor. Higher temperatures are required to eliminate aerosolsin streams with higher organic liquid composition. An additional heatedzone is used to trim the fluid temperature to control aerosols. Inaddition, this heater provides a fine level of temperature control ofthe fluid before collection in the pressurized collection chamber. Asmentioned above, a secondary effect is that a higher trim temperaturecan reduce the concentration of dissolved CO2, thereby reducing thepossibility of uncontrolled or explosive outgassing of the CO2 when thepressure is removed from the collection chamber.

Following the trim heater, a valve system is used to divert the biphasicflow stream sequentially to waste or to one of the collection chambersin a collection cassette. The valve system is comprised of one or morevalves and an electronic controller. The system is designed to offerrapid response to a manual or automated start/stop signal. Typically,the signal would result from detection of a component of interestemerging from the high-pressure flow system. A start signal would begenerated at the initial detection of the component while a stop signalwould be generated at the loss of detection. The effect of a startsignal is to divert the flow to the first unused collection chamber ofthe cassette. The effect of the stop signal is to divert the flow towaste. Another possible type of start/stop signal may be based on atimetable rather than physical detection of components. The controllermay also have features to limit the access time or flow volume allowedto an individual chamber. In addition, the controller may allow orprevent the system from cycling back to the original chamber if morefractions are desired than there exists available collection chambers.

The collection cassette is a resealable apparatus that contains one ormore hollow collection chambers open at the top. In the preferredexemplary embodiment, each chamber holds a removable inert liner. Theliner collects a fraction of the original sample dissolved in a liquidsolvent base. A preferred exemplary embodiment of a cassette has fourchambers housing four test tube vials that function as chamber liners.The number of chambers in a cassette may be varied with no effect onperformance. Each test tube vial may hold up to its capacity of aseparated sample fraction from the high-pressure flow stream.

In the preferred embodiment, sample fractions are collected in onechamber of the cassette at a time. The biphasic fluid enters a chambervia a transfer line from the valve system. The tip of the transfer lineis preferentially positioned tangential to the inner wall of thecollection tube and with a slight downward angle, usually less than 45degrees from horizontal. Attached to the transfer line and suspendedinside a test tube is a guiding spring wire. The spring wire is bowedaway from the transfer line and functions as a guide for the transferline as it descends into a vial. When transfer tubing is properlyinserted into a test tube vial, the bowed section of the spring wireengages the circumferential edge of the open end of a test tube vial. Asthe tubing continues into the test tube, the spring wire compressesagainst the inner surface of the test tube vial and pushes the tubingtowards the opposite side of the vial. As a result, the angled tip ofthe transfer tubing is pressed against the inner wall of the test tubevial.

Both the organic liquid and CO2 gas follow a descending spiral pathalong the inner wall to the bottom of the collection liner. The liquidcollects at this point and begins to fill the liner. The CO2 gascontinues in a path up the center of the liner to a vent in thecollection chamber. A restrictive transfer line attached to the ventcauses the CO2 gas to pressurize the collection chamber both inside andoutside the collection liner. The degree of back pressurization withinthe chamber is roughly proportional to the composition of CO2 in theoriginal mobile phase.

The pressurization of the collection chamber serves to slow down thevelocity of the CO2 entering the chamber. This in turn reduces themagnitude of shear forces occurring between the CO2 gas and thecollected liquid at the bottom of the liner. With lower shear forces,there is less tendency for the collected liquid to become an aerosol andto be removed from the collection tube with the exiting gas. A similareffect is obtained by the proper angling the inlet transfer linerelative to the collection tube wall. The closer the angle of the tubeis to horizontal the lower the observed turbulence at the liquidsurface. However, enough angle must be provided to insure the majorityof effluent is directed downward rather than upward on the liner wall.The two effects of back pressure and delivery angle combine to reduceaerosol formation in the collected liquid fraction. The success ofoptimizing these effects determines how close the inlet tube can come tothe collection liquid, and thereby determining how high the liner may befilled before sample loss becomes a problem. When flow to the chamber isstopped, the chamber depressurizes. Once the sample chamber isdepressurized, the liner may be removed by opening the top lid of thecassette.

The collection of fractions into disposable liners of collectionchambers may be automated through the use of robotics. An automatedsystem enables rapid substitution of test tube vials into and out ofcollection chambers and long unattended run times based on a quantity ofvials available for substitution. A programmable robot automaticallysequences cassettes between sample injections, thereby speeding up theprocess while reducing the margin for error. The automated system cancollect on the order of thousands of fractions per month.

The automated system is contained in laboratory grade housing. Thesystem is comprised of a robotic arm, a supply of test tube vialsarranged upright in racks, and an automated version of a cassetteassembly. In addition, the system may contain sufficient probes, valvesand sample containers to achieve automated delivery of unfractionatedsamples into the chromatographic or extraction system.

The collection cassette and its automated mechanisms are designed forrapid sample collection and minimal stop time between chamber linerreplacements. The cassette in the preferred embodiment has two banks offour collection chambers each. A lid is positioned above one bank ofcollection chambers in the cassette. The lid has four partially recessedannular bores corresponding to the four collection chambers in thecassette. The lid raises and lowers with action from pneumatic actuatorsmounted on the base of the housing and located on opposite longitudinalends of the lid. As the actuators simultaneously lower the lid onto thecollection cassette, the top edge of each chamber engages the bottomedges of the lid corresponding to the rims of each partially recessedbore. The lid and chambers engage and form pressure tight seals in eachchamber in preparation for sample fraction collection. The lid hastransfer and waste line tubing passing through each recessed bore thatcorrespond to each collection chamber. Each tubing pair enters a testtube as the lid is lowered onto the cassette. The spring wire attachedto the inlet tubing guides an inlet tube into a test tube vial. Anangled tip on the tube is forced against the inner wall of the testtube. After the lid has sealed on the row of collection chambers, avalve system dispenses the flowstream containing gaseous and liquidphases into the chamber liners from the sample fractionation process.

When all test tube vials in the pressurized cassette row have beenfilled and depressurized, the lid lifts off of the cassette. Thecassette then moves laterally, or shuttles, until a row containing emptycollection chamber liners is moved under the lid in place of the formerrow. The cassette is constrained to shuttle laterally along a path onthe base of the housing. The lid lowers and engages the new row ofchambers, thereby preparing the test tubes to accept sample fractions.Meanwhile, the former row of chamber liner test tube vials containingliquid fractions are removed from the collection chambers andtransported to open spaces in a storage tray via a robot arm.

In summary, samples in the preferred embodiment are dissolved in aminimum volume of modifier solvent and are collected in removable andreusable liners. Through controlling flowrate, velocity, temperature,and pressure in the system, superior separation of near-supercriticalelution fluid is obtained. Collection efficiencies of up to 98% ofinjected sample components may be realized. The cassette, by utilizingpressurized collection chambers and disposable liners in the process,minimizes the use of additional collection and cleaning solvent spent bya laboratory, which is economical and good for the environment.Laboratories and research facilities that demand purity of samples whilemaximizing output and minimizing waste will benefit from the proposedinvention. Large-scale sample fractionation and collection, numbering inthe thousands of samples per month, may be realized from the exemplaryembodiment.

BRIEF DESCRIPTION OF THE DRAWINGS

For a better understanding of the nature of the present invention,reference is had to the following figures and detailed description,wherein like elements are accorded like reference numerals, and wherein:

FIG. 1 illustrates a schematic flow diagram of the supercritical fluidchromatography system and the collection system including the samplecassette embodied in the invention.

FIG. 2 illustrates an exploded isometric view of a sample collectioncassette.

FIG. 3A and 3B illustrate top and bottom plan views of the cassette lid.

FIG. 4 illustrates a plan view of an alternative exemplary embodiment ofan automated fraction collection system.

FIG. 5 illustrates a side view of an alternative exemplary embodiment ofan automated fraction collection system.

FIG. 6 illustrates an exploded isometric view of a shuttle samplecollection cassette, lid, and mechanized controlled movement system.

FIG. 7 illustrates a detailed side view of the shuttle cassette andassociated mechanical control apparatus.

FIGS. 8A and 8B illustrate detailed cross sectional views of transfertubing before and after insertion into a test tube vial.

FIG. 9 illustrates an alternative embodiment of an integrated collectioncassette having multiple rows of collection chambers.

FIG. 10 illustrates an additional alternative embodiment of a shuttlecollection cassette for an automated system.

DETAILED DESCRIPTION OF PREFERRED EXEMPLARY EMBODIMENTS

The preferred embodiment of the apparatus is illustrated in the flowchart of FIG. 1 within the perimeter line 10. Except where noted,specifications for a preferred exemplary embodiment are given for asystem that accepts flows of 20 to 100 mL/min total flow (CO2 plusmodifier flow) in the highly compressed state from the pumping system.Flowrates for alternative embodiments could range in orders of magnitudehigher or lower through adjustment or substitution of system hardwareand flow parameters.

In the preferred exemplary embodiment, the SFC collection system iscomposed of a moderately restrictive, thermally regulated transfer tube12 which extends from a back pressure regulator 14 into a multi-portdistribution valve 22 and from the valve to a variety of flowpathsleading either through discrete collection chambers 32 or directlyconnected to a vented common waste container 26.

Expanded elution fluid leaves the backpressure regulator 14 at avelocity of approximately two to five times the flow velocity upstreamof the backpressure regulator 14 and under back pressure ofapproximately twenty to forty bars. Variations in the expansion occur asa result of the changing modifier solvent concentration from 2.5 to 50percent over the course of a separation.

Initial separation of the liquid phase sample from carbon dioxide gasoccurs immediately at the point of initial decompression within thebackpressure regulator 14 of the SFC or SFE system. By providingdownstream restriction, a minimum backpressure sufficient to prevent theformation of solid CO2 can be maintained while liquid CO2 is present inthe transfer lines 12. The degree of CO2 evaporation is a function ofboth the available heat transfer in this region and the downstream flowrestriction which limits the amount of expansion available to thedecompressing fluid. Due to the pressure drop across the backpressureregulator 14, a fraction of the emerging CO2 will evaporate, typicallycausing a significant drop in the temperature of the emerging fluid.

Further separation and evaporation of CO2 from the organic modifieroccurs in stainless steel transfer tubing 12 running between the firstbackpressure regulator 14 and the cassette 24. The transfer tubing 12containing a flowstream of the biphasic CO2 and modifier is exposed to aseries of a heaters 16, 18 designed to optimize thermal transfer to thebiphasic fluid in the flowstream. Ideally, this heater series transferssufficient energy to the liquid CO2 portion of the emerging fluid toallow for complete evaporation of the liquid CO2 and raises the fluidtemperature sufficiently to prevent ice from forming externally on thetransfer tubing 12.

During the CO2 evaporation process within the first heated zone,significant separation between the gaseous CO2 and liquid modifieroccurs. However, the separation to pure CO2 and pure organic modifier isnever realized. Some organic modifier is typically evaporated into thegas state. The degree of evaporation is largely dependent on theabsolute temperature of the fluids within the transfer tubing 12. Whileorganic modifier evaporation does lead to lower recovery of liquid phasewhen it reaches the collection cassette 24, it does not necessarilyreduce the recovery of dissolved components of interest which do nottypically have low enough boiling points to convert to vapor. A fractionof CO2 will also remain dissolved in the organic liquid modifier. Bothtemperature and pressure determine the amount of residual CO2. Highertemperatures reduce CO2 solubility while higher pressures increase CO2solubility. Turbulent flow of the CO2 gas within the narrow tubing alsoproduces a strong shearing force that propels the liquid down the wallsof the transfer tube 12. This very turbulent flow frequently causessmall droplets at the liquid surface to rip away from the bulk liquidand become entrained into the rapidly moving gas phase of the fluid downthe transfer tube 12. Such an effect is called aerosol formation, or“misting”.

A plurality of heaters may be mounted in series to heat the elutionfluid. In FIG. 1, the preferred exemplary embodiment has an evaporatorheater 18 and a trim heater 20 mounted in series after the backpressureregulator 14. The evaporator 18 is heated with an appropriately sizedcartridge heater and controlled by an appropriate heater controller. Inthe preferred embodiment, transfer tubing 12 is tightly coiled aroundthe heating assembly and optimized for thermal contact. The elutionfluid is heated to within the control temperature of the evaporator 18,which is between approximately 5 to 50 degrees C., to protect heatsensitive compounds from being damaged. The objective is to boil CO2 outof the elution fluid as the fluid passes through the evaporator 18. Tocomplete the required heat transfer, biphasic elution fluid insidetransfer tubing 12 enters the final heat exchanger, which is a trimheater 20. In the preferred embodiment, the trim heater setting istypically above the evaporator 18 setpoint. The heater 20 is used notonly to suppress aerosol formation within the transfer tube 12 but alsoto control the level of dissolved CO2 in the liquid phase.

It is beneficial to slow the velocity of fluids within the transfertubing 12 passing through the heater series 18,20. The fluid velocity isslowed inside the transfer tubing 12 by placing a restrictive orifice orsmaller diameter tube immediately downstream from first heater series.Elution fluid exits the evaporator 18 and enters a flow restrictor 16,which provides a higher backpressure in the evaporator 18 and therebyslows the flow and increases the contact time of the liquid CO2 phase.The restrictor 16 also insures a high enough backpressure to prevent theliquid carbon dioxide from forming solid carbon dioxide, also known asdry ice. The restriction increases the backpressure in the heated zoneand reduces the amount of the gas expansion. In an alternative exemplaryembodiment, the velocity of fluids can be slowed after all heaters,however such a configuration does not control the final expansion of CO2which can result in uncontrolled cooling of fluids within the transferlines. As a result, the ability to actively suppress aerosol formationmay be diminished.

After exiting the trim heater 20, transfer tubing 12 connects to thecommon port of a valve system 22. The valve system in the preferredexemplary embodiment is a multi-port selector valve 22. As elution fluidfrom the peak of interest passes through the valve system 22, the gasand liquid phases are directed into either a collection cassette 24 orto a waste stream container 26. The outlet ports on a multi-portselection valve 22 are connected to a plurality of transfer tubing lines28. The transfer lines 28 pass through a cassette lid 30 and intodiscreet chambers 32 within the cassette 24. The transfer lines 28 haveairtight and pressure resistant connections into and out of the cassettelid 30. The remaining ports in a multi-port selection valve 22 connectto waste transfer lines 34. In an alternative exemplary embodiment,multiple discreet valves are installed and connected to the incomingtransfer line 12, having each valve port connected to an individualcollection chamber 32 in the cassette 24 and a discreet valve connectedto a waste line 34.

Inlet lines 28 entering a collection chamber 32 insert into a test tubevial 36 within a chamber 32. Liquid phase 38 is captured in a test tube36 while gaseous phase escapes out of a chamber 32 through a dischargeline 40. Gas in the discharge line 40 is flowing at high pressure.Discharge lines 40 from the cassette 24 run through a pressure reliefswitch 42 to protect the cassette and upstream components from possibledamage due to over-pressurization from a system malfunction.

Referring additionally to FIG. 2, a preferred exemplary embodiment ofthe cassette 24 comprises four discreet collection chambers 32. However,in alternative embodiments, one or more individual chambers 32 arepossible in the cassette 24. Each collection chamber 32 in the preferredembodiment is a closed system that is the final separation point ofliquid and gaseous phases. Chambers 32 are hollow cylinders constructedof high strength transparent plastic to allow visual monitoring ofseparation and collection processes. The cassette chambers 32 can beformed of stainless steel or other appropriate laboratory-gradematerials. The chambers 32 sit parallel and upright in the cassette 24.Each chamber 32 is constrained at its upper and lower ends within amolded frame 44, 46. Each chamber 32 is set with the open end surroundedby the upper molded frame 44 and the lower end partially embedded intothe lower molded frame 46. Communication of liquid or gaseous phasesbetween chambers 32 is prohibited by seals 48 that are seated in agroove 50 at the top, open end of each chamber 32.

Each collection chamber 32 houses a removable, replaceable liner. Astandard glass test tube vial 36 functions as a liner and is seatedupright inside each the chamber 32. The closed bottom of a test tubevial 36 rests on the base of the chamber 32 and is easily removable.Once inserted, the top of the test tube vial 36 must be lower than thecombined height of a chamber 32 and the internal recessed bore 60 (FIG.3) of a lid piece 30 when the lid and cassette 24 are engaged. A testtube vial 36 and a chamber 32 are a single pressurized system thatcommunicate through the top of the chamber 32. The test tube vial 36functions as a disposable liner for the chamber 32 to capture the liquidphase 38 that has separated from the flow stream. The inside of the vial36 and the annular space of the chamber 32 surrounding the vial areequilibrated to the same pressure, which is a range of approximately 20to 100 psig during separation processes for a flowstream up to 50ml/min. This arrangement enables sample fraction collection at highpressure using standard laboratory glass test tube vials 36 without arisk of breaking the glass vial inside the chamber 32.

FIG. 2 illustrates the cassette 24, comprising a rectangular framesecuring four upright chambers 32. The upper section 44 and lowersection 46 of the molded frame hold the chambers 32 in place. The frameis completed by two rigid rectangular end pieces 52 attached to theupper and lower sections. Each end piece 52 is a metal plate fastened tothe upper 44 and lower 46 frame sections with machine screws 54.Butterfly latches 56 are installed at the top of both rigid end pieces52 secure the lid piece 30 to the top of the cassette 26. The lid 30 maybe removed manually between sample injections for quick access to, andremoval of, chamber liners 36. As illustrated in FIG. 1, the bottom ofeach chamber 32 has a transfer tube or orifice 33 running completelythrough the base of a collection chamber and lower frame 46. The orifice33 through the chamber base 46 can be used to remove liquid phase fluidfrom a chamber 32 without opening the chamber or depressurizing thechamber 32. The sample discharge port 33 also permits easier drainingand cleaning of the chamber 32 during maintenance of the cassette 24.

FIGS. 3A and 3B illustrate top and bottom views of the removablecassette lid 30, respectively. The lid 30 has four sets of threeboreholes 58 in a triangulated pattern positioned such that each set ofboreholes is directly over each of the chambers 32 when the lid 30 isengaged to the cassette 24. The bottom face of the lid 30 has partiallyrecessed bores 60 positioned directly above each chamber 32 when the lid30 and cassette 24 are engaged. The diameter of a recessed bore 60 issized slightly smaller than chamber 32 diameters. The recessed bore's 60perimeter is positioned completely inside of a seal 48 when the lid 30is fastened to the cassette, as illustrated in FIG. 2. The recessedboreholes 60 allow a test tube 36 to stand taller than the top planarsurface of the upper frame section 44 of the cassette 24 so that a testtube 36 may be removed without reaching into a collection chamber 32,thereby possibly cross-contaminating subsequent samples. To guide thelid 30 and cassette base 24 together when engaging, alignment pins 62,illustrated in FIG.3, are formed on the outer, top surface of thecassette frame 24. Partially recessed bores 63 in the lid 30 receive thealignment pins 62 from the cassette frame 24. Catches 64 for thebutterfly latches 56 are attached to each long end of the lid 30.

Inlet transfer tubing 28 carries liquid and gaseous phases into testtube vials 36 housed in each collection chamber 32 of the cassette. Eachinlet tube 28 fits through a hole 58 in the lid 30 and inserts into atest tube vial 36. Proper fittings on the tubing 28 provide airtightconnections that can also withstand pressure forces in the SFC system.Inlet tubing probes 66 direct elution fluid into a test tube vial 36 andan outlet tube 68 provides an escape route for gas that is underpressure to exit the chamber 32 and discharge to waste collection 26.

In the preferred embodiment, fractions are collected in one chamber 32of the cassette 24 at a time. During the fractionation process, both theliquid phase and the gas phase discharge into the collection vial 36where final separation takes place. The pressurization of the collectionchamber 32 serves to slow down the velocity the CO2 within the chamber32. This in turn reduces the magnitude of shear forces occurring betweenthe CO2 gas and the collected liquid at the bottom of the liner 36. Withlower shear forces, there is less tendency for the collected liquid tobecome an aerosol and removed from the collection liner 36 with theexiting gas. A similar effect is obtained by the proper angling theinlet transfer line relative to the collection liner 36 wall. The closerthe angle of the tube 66 is to horizontal the lower the observedturbulence at the liquid surface. However, enough angle must be providedto insure the majority of effluent is directed downward rather thanupward on the liner 36 wall.

The biphasic elution fluid enters a chamber 32 via a transfer line 28from the valve system 22. As illustrated in FIGS. 8A and 8B, the tip ofthe transfer tube 66 is a probe preferentially positioned tangential tothe inner wall of the collection vial 36 and with a slight downwardangle, usually less than 45 degrees from horizontal. Attached to theprobe 66 is a guiding spring wire 70. The spring wire 70 is bowed awayfrom the probe 66. The spring wire 70 acts as a guide for the probe 66as the probe descends into a test tube vial 36. When the probe 66 isproperly inserted into a test tube vial 36, the bowed section of thespring wire 70 contacts the circumferential edge of the open end of atest tube vial 36. As the tubing 66 continues into the test tube vial36, the spring wire 70 compresses against the inner surface of the vial36 and pushes the probe 66 towards the opposite side of the vial 36. Asa result, the angled tip of the probe 66 is pressed against the innerwall of the test tube vial 36.

The spring wire 70 is extruded from inert materials that will notchemically interfere with collected samples in the test tube vials 36.In an alternative exemplary embodiment, the probe section 66 of thetransfer tubing 28 is a rigidly held stainless steel probe attached tothe cassette lid 30. Metal versions of probe 66 may be terminated with alarger OD Teflon tube sleeved onto the metal probe to prevent scratchingand possible rupture of the inner wall of the collection liner 36.

Both the organic liquid and CO2 gas follow a descending spiral pathalong the inner wall to the bottom of the collection liner 36. Theliquid phase collects at this point and begins to fill the test tubevial 36. The CO2 gas continues in a path up the center of the vial 36 toa vent through the top of the collection chamber 32. A restrictivetransfer line attached 72 to the vent causes the CO2 gas to pressurizethe collection chamber 32 both inside and surrounding the collectionliner 36. The degree of back pressurization within the chamber isroughly proportional to the composition of CO2 in the original mobilephase.

The two effects of back pressure and delivery angle combine to reduceaerosol formation in the collected liquid fraction. The success ofoptimizing these effects determines how close the inlet tube 66 can cometo the collection liquid, and thereby determining how high the liners 36may fill before sample loss becomes a problem. When flow to the chamber32 is stopped, the chamber depressurizes. Once a chamber 32 isde-pressurized, the test tube vial 36 containing liquid phase may beremoved by opening the top lid 30 of the cassette 24.

The outlet line tubing 72 from each chamber 32 is connected to a fixedrestrictor 42 to keep pressure inside the chambers 32. The fixedrestrictor 42 raises the upstream pressure between approximately 20 and100 psig depending on CO2 flow rate. Each discharge line 72 passesthrough a pressure switch 78 to protect against overpressuring andrupturing. Pressure in each chamber is monitored visually with apressure gauge 76 that is threaded into the lid 58 over each chamber 32.Discharge lines 72 are directed to a waste collection tank 26, fromwhich the CO2 is vented. To increase laboratory safety, the systemshould not have any exposure of waste effluent, samples, or vented CO2to ambient laboratory air. The liquids and gasses in the system remainin a contained system that can be directed to a hood or safety exhaust26 to maximize safety for the technician.

The volume of the captured fractionated liquid phase 38 in thecollection vial 36 is controlled manually or automatically. Automaticcontrol in the preferred exemplary embodiment of the valve system 22 andis comprised of one or more valves and an electronic controller. Thevalve system 22 is designed to offer rapid response to a manual orautomated start/stop signal. A signal can result from detection of adetection of a component of interest emerging from the high pressureflow system. A start signal would be generated at the initial detectionof the component while a stop signal would be generated at the loss ofdetection. The effect of the stop signal is to divert the flow to wastelines 26 or to another chamber 32. An alternative embodiment of a typeof start/stop signal may be based on a time-table rather than physicaldetection of components. The controller may also have features to limitthe access time or flow volume allowed to an individual chamber 32. Inaddition, the controller may allow or prevent the system from cyclingback to the original chamber 32 if more fractions are desired than thereexist available collection chambers 32.

An alternative exemplary embodiment of the collection cassette andsystem is illustrated in FIGS. 4 through 7. This embodiment is anautomated system that utilizes a robotic arm 80 to replace chambercollection liners 36 after filling with sample fractions. Therobotically controlled unit is designed for rapid filling andreplacement of chamber liners 36 combined with a long unattended runtime. Supply trays 86 of clean test tube vials 36 that function aschamber liners 36 are located within the unit's housing 82. A roboticarm 80 is controlled to replace one or more liners 36 from a row ofcollection chambers 32 in a collection cassette 84 with liners 36 from afresh supply rack 86. The robotic arm 80 is mechanized to replace liners36 on a first row of the cassette 84 while liners 36 on a second row areautomatically moved into place. This robotically automated alternativeembodiment provides faster sample collection through a minimum of downtime to replace liners 36 as well as the ability to collect a greaternumber of samples during an unattended session.

FIGS. 4 and 5 illustrate the plan and side views, respectively, of anautomated alternative exemplary embodiment of the SFC sample collectionsystem. The components for the system are partially enclosed with alaboratory-grade housing structure 82 having a raised mounting base 88within the housing 82. The housing 82 is supported with adjustable feet90 that are distributed around the base of the housing 82. The feet 90adjust the level the housing 82 to compensate for uneven or slantedsurfaces. Supplies of uncontaminated test tube vials 36 are stored inracks 86 placed on a raised interior base 88 of the housing 82. Eachtest tube vial 36 is held upright and secured in-place in a rack 86 bymolded supports. Each support rack 86 consists of circular sectionsattached tangentially to neighboring sections, forming multiple rows andcolumns. The molded supports loosely secure test tube vials 36 that areheld in each circular opening of the racks 86. The vials 36 aremaintained equidistant from each neighboring vial to provide adequatespacing for a grabbing jaw 92 on a robotic arm 80 to grasp a vial 36without interference from a neighboring vial. The spacing also preventschipping or breakage during movement and replacement of the rack 86. Tworacks 86 of test tube vials 36 are illustrated in the Figures, howeverthe system could easily expand to a plurality of racks of the vials 36.

An alternative exemplary embodiment of a cassette 84 and associatedsystem devices is installed on the raised interior base 88. The cassette84 has a plurality of rows of chambers that are constrained to lateralmovements that are automatically controlled with a pneumatic actuator96. This cassette 84 is termed the “shuttle cassette”, or simply “theshuttle.” FIGS. 6 and 7 illustrate the shuttle cassette 84 in isometricand side views, respectively. The shuttle cassette 84 is constructedsimilar to the exemplary embodiment with an added row of collectionchambers 102. The shuttle 84 comprises upper and lower rectangularmolded frames 98, 100 supporting a plurality of rows of uprightcylindrical collection chambers 102. The shuttle 84 is constructed withtwo rows of four cylindrical collection chambers 102 in each row. Thesize of the shuttle 82 can be modified to add additional rows ofchambers 102 or additional chambers per row, such as an alternativeembodiment featuring three rows of chambers 102 illustrated in FIG. 10.The shuttle cassette 84 is formed on two opposite ends with rigidrectangular plates 104. Each end plate 104 is fastened to the upper 98and lower 100 molded frame sections with machine screws 106. The shuttle84 may be constructed with permanent attachments and fittings, however,a shuttle that readily disassembles allows easier and thorough cleaningand replacement of worn or damaged components.

The collection chambers 102 are formed of high-strength transparentplastic, which allows visual monitoring of the collection process insideof each chamber 102. As an alternative, the chambers 102 may be formedof stainless steel or a similar high-strength material compatible withSFC parameters described herein. Each cylindrical chamber 102 is setinto the lower molded frame 100 for base support. The upper molded framesection 98 is secured near the open, top end of each chamber 102. Eachchamber 102 extends above the top surface of the shuttle 84 at astandardized distance adequate to seal the chambers 102 with anautomated lid piece 108. Standard laboratory test tube vials 36 may beinserted into each of the chambers 102 to act as a removable ordisposable liner for each chamber.

The automated shuttle cassette 84 is constrained to lateral movements onthe inner raised base 88. The lower molded frame section 100, or base,of the shuttle cassette 84 has an horizontally bored hole 110,illustrated in FIG. 7, running perpendicular to the open sides of theshuttle. Offset from the shuttle 84 is an actuator 96 installed on theraised base 88 of the housing unit 82. Attached to the actuator 96 isrod 94 or controller arm. The rod 94 is constructed of a rigid material,such as stainless steel, and inserts into the bored hole 110 in the baseof the shuttle cassette 84, wherein it is firmly attached to the baseframe 100. The actuator 96 executes lateral movements of the shuttle 84according to commands sent from a programmable control system. In analternative embodiment, the base of the shuttle 100 has small rollers112 installed around the base, as illustrated on FIG. 7. The rollers 112are guided laterally by grooved tracks in the base of the housing 88.The tracks not only constrain the movement of the shuttle 84 but alsoremove tension from the controller arm 94 and actuator 96 gears causedby the shuttle 84 drifting into angled movements caused by unevenfriction on the rollers, initial off-center displacement after shuttle84 installation, or irregularities on the surface of the housing base88. Other methods of providing constrained lateral movement are possiblein alternative embodiments, such as utilizing guide tracks whereinguides on the shuttle 84 are enclosed within tracks riding onball-bearings.

Referring to FIGS. 6 and 7, the lid 108 of the shuttle cassette 84 isautomatically controlled to engage a row of collection chambers 102after the shuttle is moved into place directly below the lid 108 by thelateral actuator 96. In the alternative embodiment, the lid 108 isconstructed of stainless steel. However, high density plastic, or asimilar material having equivalent rigidity and composition for use inthe collection system, is sufficient. The lid 108 has a hole 114 througheach longitudinal end, bored parallel to the vertical axis of the lid.The holes 114 in each end of the lid 108 are sized to fit a threaded rod116. Two nuts 118 threaded above and below the lid 108 secure the lid toeach rod 116. The lid 108 is constrained to move only in the verticalplane. The movements of each rod 116 are controlled by actuators 120mounted to the raised base of the housing 88. The two pneumaticactuators 120 controlling the lid movements are synchronized to move therods 116 vertically, thereby raising and lowering the lid 108 onto a rowof collection chambers 102 in the shuttle cassette 84.

FIG. 7 illustrates the lid piece 108 raised above the shuttle 84 priorto engagement. The bottom face of the lid 108 has four bores 122partially recessed into the lid corresponding to four chambers 102 in arow of the shuttle. As the lid 108 is lowered by the pneumatic actuatorsonto the shuttle 84, each chamber 102 of a row partially inserts into arecessed borehole 122. The lid 108 stops at a programmed point at whichthe circular edge of each bore 122 engages and seals against the flatupper surface of the shuttle frame 98. Each partially recessed borehole122 in the lid 108 has a diameter larger than the chamber's 102diameter. As the lid 108 lowers onto the shuttle 84, the recessedboreholes 122 are lined up with the top, open ends of the chambers 102.The larger diameter recessed boreholes 122 each totally enclose the openend of each chamber 102. An appropriate sealing O-ring or similarcomponent is placed around the top of each chamber 102, between the topof the shuttle 84 and the lid 108, to provide an airtight and pressureresistant seal when the two components engage. Alignment pins 124 arelocated on the top surface 98 of a shuttle 84 at both ends of each rowof chambers 102. The pins 124 are shaped as half-spheres on the topsurface of the shuttle 84 and provide additional protection for shuttlecollection chambers 102 from misalignment of the shuttle 84 to the lid108. As the lid 108 engages onto the shuttle 84, the alignment pinsengage corresponding bores 126 in the lid.

A collection chamber 102 is a discreet system that is the finalseparation point of liquid and gaseous phases. Communication of liquidor gaseous phases between chambers 102 is prohibited through the lid 108that seals each chamber airtight as it automatically lowers onto a rowof chambers in the shuttle cassette 84. Similar to the exemplaryembodiment of the cassette, each chamber 102 in the shuttle 84 holds achamber liner 36 to catch fractionated liquid phase. The liner 36 is astandard laboratory test tube vial 36. The closed bottom of the testtube 36 rests at the base of each chamber 102, which rests on the lowermolded frame of a shuttle 100. A test tube vial 36 and chamber 102communicate as a single pressurized system. FIG. 8B illustrates theposition of the open end of a vertically disposed test tube vial 36below the top of a recessed borehole 122 after the lid 108 engages theshuttle 84. The inner pressure of the test tube vial 36 and thechamber's 102 annular space surrounding the vial are equilibrated andrange from approximately 20 to 100 psig during collection processes.This arrangement enables sample fraction collection at high pressureusing standard lower pressure glass or plastic vials by equilibratingthe pressure forces inside and outside the vial 36.

As illustrated in FIG. 7, the lower, closed end of each chamber 102 hasa sample discharge port 128 running completely through the lower shuttleframe 100. A plug is inserted into each sample discharge port 128 duringregular use of the shuttle 84. The sample discharge port 128 permitsremoval of liquid phase that is collected directly into a chamber 102without using a liner. By withdrawing liquid phase through the sampledischarge port 128, the liquid phase may be collected withoutdisengaging the lid 104 from the shuttle 84. Liquid phase may beevacuated from a chamber 102 under pressure or gravity fed out of achamber after chamber depressurization.

Inlet 66 and outlet 68 tubing for transferring influent and effluentliquid and gas phases between the shuttle cassette 84 and externaltransfer lines are illustrated in FIGS. 6 and 7. Inlet 66 and outlet 68tubing for the shuttle 84 pass through the lid 108. Transfer tubing66,68 is constructed from high-pressure stainless steel or equivalentmaterials. Inlet tubes 66 carry gaseous and liquid phases into acollection chamber 102 under high pressure. Outlet tubes 68 carryseparated gaseous phase to a waste tank 26 for venting or disposal. Thelid section 108 has four sets of three holes 134 in triangularformations that pass through the lid and are located to correspond withcollection chambers 102 when the lid is engaged to the shuttle cassette84.

In addition to transfer tubing, one of the holes 134 permits measurementof pressure forces inside a chamber with a pressure gauge 76 threadedinto the hole 134 from top of the lid 108. The transfer tubing 66, 68and pressure gauge 136 all have pressure resistant airtight fittingsspecified to withstand pressure forces created in the SFC system.Transfer tubes 66, 68 installed below the lid 108 insert into a testtube vial 36 when the lid 108 is engaged to the shuttle cassette 84. Thetip of each inlet tube 66, or probe, is constrained to an angle lessthan 45 degrees and wrapped with non-reactive spring wire 70 that isbowed along the vertical section, similar in construction and purpose asdescribed in the preferred embodiment. The spring wire 70 serves toangle the inlet tubing 66 inside a test tube vial 36 by applyingpressure forces against the vial's 36 inner wall. As a result, the opentip of the inlet tube 66 is forced tangentially against an opposinginner wall of the vial 36. This configuration of the inlet tube 66 isdesirable because it causes the liquid phase that exits the inlet tube66 to contact a side wall of the vial 36 and swirl down the inner wallof the vial 36 in a spiraling motion. The swirling action provides thefinal separation process of liquid phase from entrained gaseous phasewhile preventing re-entrainment and loss of sample fractions from theliquid phases into gaseous phases or aerosol mists that can be carriedaway with gaseous phases to a waste vent 26.

In an alternative exemplary embodiment, a robotic arm, such as aCartesian or three-dimensional robotic arm, is programmably controlledto move test tube vials between supply racks and the shuttle cassettecollection chambers. FIGS. 4 and 5 illustrate a three-dimensionalrobotic arm 80 mounted to a wall of the unit housing 82 near the shuttlecassette 84. A host PC or microcontroller issues positioning commandsfor the arm's movement and controls automated functions. The arm 80 hasa jaw 92 to grab and place test tube vials 36 into the shuttle cassette84 from the test tube supply racks 86. The jaw 92 is controlled to griptest tube vials 36 of specific outer diameter and at specific locationswithin the unit 82. In the alternative embodiment illustrated in FIG. 5,the robotic arm 80 is gripping one test tube 36 in its jaw 92 to movethe test tube between the shuttle 84 and a supply rack 86. To increasethe volume of vials 36 exchanged, the gripper jaw 92 could be modifiedto grip two or more test tube vials, multiple jaws could be placed on asingle arm 80, or multiple robotic arms could work on the sameembodiment. The arm 80 acts in concert with the automated movements ofthe shuttle 84. As a row of chambers 102 in the shuttle 84 is engaged tothe lid 108, the robotic arm 80 replaces test tube vials 36 in theshuttle that are filled with collected sample fractions with fresh vials36 from a supply rack 86. When a row of test tubes 36 in the shuttle 84have been replaced, and the row of vials 36 under the lid 108 havecaptured liquid phase fractions, a programmable controller signals thepneumatic actuators controlling the lid 120 to disengage and move thelid 108 away from the shuttle 84. The lateral control 96 of the shuttle84 is then signaled to move the shuttle such that the row of chambers102 containing clean, uncontaminated test tube vials 36 correspond to aposition underneath the lid 108 prior to engagement. The lid actuators120 are then signaled to engage the lid 108 again to the shuttle 84,thereby preparing the chambers to receive liquid phase fractions. Therobotic arm 80 next grabs vials 36 from the exposed shuttle chambers 102that contain liquid phase fractions and places them into a supply rack86. The arm 80 then replaces an uncontaminated vial 36 into each emptychamber 102 until a row of chambers is completely filled with fresh testtubes. This process is repeated for the length of a sample run or untilthe system is depleted of uncontaminated test tube vials from the supplyracks 86.

An alternative embodiment of a collection cassette is illustrated inFIG. 9. An integrated cassette 140 consists of multiple rows of wells144 in a grid pattern formed similar to a titration tray. The smallerfootprint of the integrated cassette 140 can increase the density ofcollection chambers over the shuttle cassette 84. The integratedcassette 140 also functions as a storage tray for gathered liquid phasefractions. Therefore, time and expense are saved during samplingprocedures by removing the steps of the substituting chamber liners 36and replacing liners from a separate storage area. By modifying the lid108 and mechanics of the automated collection system, the integratedcassette 140 may serve as its own sample collection cassette and storagetray and can rapidly receive fractions without having to replace liners36 between each sample injection. The robotic arm 80 in the system mayreplace integrated cassette 140 units as a whole after a sampling eventis completed or chamber wells 144 contain the desired amount of liquidphase fractions. A plurality of integrated cassettes 140 are stored inthe automated collection system providing the means for hundreds ofcollected fractions during an automated run. A preferred construction ofan integrated cassette is a 4×6 chamber array in the deep-well microtiter plate format used commonly in the pharmaceutical industry. Such aformat improves automation storage density not only due to more chambersper area, but these chambers are also easily stackable, which gives anadded dimension of sample storage capacity. This alternative embodimentis a shuttle cassette tray 140 formed from high-strength materials suchas plastic, resin, or stainless steel.

The integrated cassette tray 140 is also advantageous for rapid fractioncollection because it can be modified to contain replaceable liners 36in the wells 144 or use no liners, thereby collection liquid fractionsdirectly into the wells 144. The integrated cassette 140 can be replacedas a unit after wells 144 are filled with liquid phase fractions.

An alternative embodiment of an automated system using a cassette traywould appear similar to that illustrated in FIG. 4 but with certainmodifications. Modifications to the automated system include spacing fora supply of cassette trays 40 instead of test tube racks 86, sizing ofthe lid piece 108 and associated mechanized controllers 120 and transfertubing 66, 68, sizing of lateral mechanized controllers 96 for the tray140 while switching between rows of chambers 144 during fractioncollections, and modification of a robotic arm 80 to substitute filledcassette trays 140 with new trays from a supply area. An alternative tothis configuration is having a moveable lid section 108 connected to arobotic arm 80 that engages each row of chambers in a supply rack oftrays 140 without ever moving the trays.

As can be understood from the above description, the sample collectionsystem has several advantages, for example: it provides simplifiedprep-SFC sample collection; it collects only fractions of interest fromthe injected sample; it collects purified samples into removable,inexpensive, and disposable collection vials; it provides extremelyefficient and controllable gas and liquid phase separation, therebyproviding up to 98% consistent sample recovery; it is environmentallyfriendly and economical because it eliminates additional use of solventsto collect, trap, or recover samples, and clean unnecessary associatedmechanical separation equipment; it allows high speed, high volume, andhigh purity SFC sample collection.

Because many varying and different embodiments may be made within thescope of the inventive concept herein taught, and because manymodifications may be made in the embodiments herein detailed inaccordance with the descriptive requirements of the law, it is to beunderstood that the details herein are to be interpreted as illustrativeand not in a limiting sense.

What is claimed:
 1. A process for collecting samples from a flow streamcontaining a mixture of highly compressed gas, compressible liquid orsupercritical fluid and a relatively incompressible liquid, comprisingthe steps of: controlling the pressure, temperature and velocity of saidflow stream to enhance separation processes of a monophasic fluidmixture into separate gaseous and liquid phases; suppressing formationof aerosols within said separation processes; redirecting said flowstream through a valve system according to a physical starting eventinto a collection cassette having one or more collection chambers;retaining said liquid phase in said collection chamber and venting saidgaseous phase to a waste stream; redirecting said flow stream throughsaid valve system according to a physical stopping event into said wastestream or a second collection chamber.
 2. The process according to claim1, wherein: said flow stream is the effluent of a supercritical fluidchromatography system or supercritical fluid extraction system.
 3. Theprocess according to claim 1, further comprising: transfer tubingcarrying said flow stream enhances said separation processes of saidmonophasic fluid mixture into separate gaseous and liquid phases.
 4. Theprocess according to claim 3, wherein: said transfer tubing has innerdiameter of approximately 0.030 to 0.063 inches, corresponding to aflowrate in said flow stream of approximately 20 to 100 mL/min.
 5. Theprocess according to claim 1, wherein: separation of said incompressibleliquid is enhanced by controlling the temperature in said flow stream tosuppress aerosols in said collection chamber.
 6. The process accordingto claim 1, further comprising: pressure in said flow stream is loweredby the steps of adding restrictors to said flow stream to slow thelinear flow rate of said flow stream.
 7. The process according to claim1, wherein-further comprising: said physical starting and stoppingevents result from a detection device in said flow stream.
 8. Theprocess according to claim 1, wherein: said physical starting andstopping events result from a timing device.
 9. The process according toclaim 1, wherein: said physical starting and stopping events result froma manual signal.
 10. The process according to claim 1, furthercomprising: the volume and direction of said flowstream is controlledwith an automatic switching valve system.
 11. The process according toclaim 1, further comprising: the volume and direction of said flowstream through said flowstream is controlled with a manual switchingvalve system.
 12. A process according to claim 1, wherein: saidflowstream collected in said sample collection chamber is pressurecontrolled to prevent loss of liquid phase due to aerosol formation. 13.The process according to claim 1, further comprising: collecting saidliquid phase in a replaceable collection chamber liner housed withinsaid collection chamber, said liner is equilibrated to the pressureinside said collection chamber.
 14. The process according to claim 1,wherein: said flow stream discharges from said transfer tubetangentially to the inner wall of said collection chamber.
 15. Theprocess according to claim 14, further comprising: a spring wireattached to said transfer tube discharging tangentially to the innerwall of said collection chamber, said spring wire compressing againstsaid inner chamber wall and transferring pressure forces to saidtransfer tube.
 16. The process according to claim 1, further comprising:automatically resetting said collection chamber to an uncontaminatedstate by robotically replacing a collection liner housed within saidcollection chamber.
 17. The process according to claim 1, furthercomprising: automatically resetting a plurality of collection chambersintegrated as a cassette tray to an uncontaminated state by roboticallyreplacing each cassette tray during sample collection processes.
 18. Theprocess according to claim 1, further comprising: removing said liquidphase fluid from said collection chamber through a sample discharge portin said chamber.
 19. The process according to claim 1, wherein: saidcollection system repetitively collects similar fractions from differentinjected samples into the same chamber.
 20. A process for collectingsamples from a flow stream containing a mixture of highly compressedgas, compressible liquid or supercritical fluid and a relativelyincompressible liquid, comprising the steps of: controlling thepressure, temperature, and velocity of said flow stream to enhanceseparation processes of a monophasic fluid mixture into separate gaseousand liquid phases; suppressing formation of aerosols within saidseparation processes; redirecting said flow stream through a valvesystem according to a physical starting event into a collection cassettehaving one or more collection chambers; retaining said liquid phase in areplaceable collection liner housed within said collection chamber;equilibrating pressure in said collection chamber with said liner;venting said gaseous phase to a waste stream; redirecting said flowstream through a valve system according to a physical stopping eventinto said waste stream or a second collection chamber liner.
 21. Theprocess according to claim 20, wherein: said flow stream is the effluentof a supercritical fluid chromatography system or supercritical fluidextraction system.
 22. The process according to claim 20, furthercomprising: transfer tubing carrying said flow stream, said transfertubing enhancing said separation processes of a monophasic fluid mixtureinto separate gaseous and liquid phases.
 23. The process according toclaim 20, wherein: said flow stream flows through transfer tubing havinginner diameters of approximately 0.030 to 0.063 inches, corresponding toa flowrate in said flow stream of approximately 20 to 100 mL/min. 24.The process according to claim 20, wherein: collection of saidincompressible liquid is enhanced by controlling the temperature in saidflow stream to reduce aerosols in said collection chamber.
 25. Theprocess according to claim 20, further comprising: pressure in said flowstream is lowered by the steps of adding restrictors to said flow streamto slow the linear flow rate of said flow stream.
 26. The processaccording to claim 20, wherein: said physical starting and stoppingevents result from a detection device, timing device, or manual signalin said flow stream.
 27. The process according to claim 20, wherein:controlling the volume and direction of said flow stream through saidvalve system with an automatic or a manual switching valve system.
 28. Aprocess according to claim 20, wherein: said sample collection chamberis pressure controlled to prevent loss due to turbulence of said liquidphase.
 29. The process according to claim 20, further comprising:discharging said flow stream tangentially to the inner wall of saidcollection chamber through said transfer tubing.
 30. The processaccording to claim 20, further comprising: compressing a spring wirebetween said transfer tube and the inner wall of said collectionchamber, thereby discharging said flow stream tangentially to said innerchamber wall.
 31. The process according to claim 20, further comprising:automatically resetting said collection chamber to an uncontaminatedstate by robotically replacing a collection liner housed within saidcollection chamber.
 32. The process according to claim 20, furthercomprising: automatically resetting a plurality of collection chambersintegrated as a cassette tray to an uncontaminated state by roboticallyreplacing each cassette tray during sample collection processes.
 33. Theprocess according to claim 20, wherein: said collection systemrepetitively collects similar fractions from different injected sampleruns into similar said collection chambers.
 34. A process for collectingsamples from a flow stream containing a mixture of highly compressedgas, compressible liquid or supercritical fluid and a relativelyincompressible liquid, comprising the steps of: controlling thepressure, temperature and velocity of said flow stream to enhanceseparation processes of a monophasic fluid mixture into separate gaseousand liquid phases; suppressing formation of aerosols within saidseparation processes; directing said flow stream through a valve systemaccording to a physical starting event into a first collection cassettehaving one or more said collection chambers; retaining said liquid phasein said collection chamber and directing said gaseous phase to a wastestream; automatically resetting said collection chamber to anuncontaminated state; redirecting said flow stream through said valvesystem according to a physical stopping event into said waste stream ora second uncontaminated collection chamber.
 35. A process according toclaim 34, further comprising: automatically resetting said cassette toan uncontaminated state by robotically replacing a first collectionchamber liner from said cassette with a second uncontaminated collectionchamber liner.
 36. A process according to claim 34, wherein:automatically resetting said collection chamber to an uncontaminatedstate comprises the steps: disengaging said lid from said cassette;removing said liner with a robotic arm and placing said liner into astorage area; replacing said liner with said robotic arm with anuncontaminated liner; re-engaging said cassette with said moveable lid.37. A process according to claim 34, wherein: said lid movement iscontrolled with a pneumatic actuator.
 38. A process for collectingsamples from a flow stream containing a mixture of highly compressedgas, compressible liquid or supercritical fluid and a relativelyincompressible liquid, comprising the steps of: controlling thepressure, temperature and velocity of said flow stream to enhanceseparation processes of a monophasic fluid mixture into separate gaseousand liquid phases; suppressing formation of aerosols within saidseparation processes; automatically engaging a lid onto wells in acassette tray; redirecting said flow stream through a valve systemaccording to a physical starting event into wells in a cassette tray;retaining said liquid phase in said wells and venting said gaseous phaseto a waste stream; automatically disengaging said lid from said wells;automatically resetting said cassette tray to an uncontaminated state byrobotically replacing said cassette tray; redirecting said flow streamthrough said valve system according to a physical stopping event intosaid waste stream or a second cassette tray; collecting a liquid phasesample from said flow stream by robotically moving said lid between saidwells in said cassette tray.
 39. A process according to claim 38,further comprising: automatically moving a cassette away from said lidafter lid has disengaged from said cassette, at which time saidcollection chamber is robotically removing from said cassette andreplaced with a second uncontaminated collection chamber.
 40. A processaccording to claim 38, further comprising: replaceable liners housed insaid cassette tray, said liners automatically reset to an uncontaminatedstate by robotically replacing said liners within said cassette tray.